Aberrant control of gene expression programs is contributing to the pathogenesis of a number of globally significant diseases, including cardiovascular disease (CVD), obesity, and diabetes. Members of the sterol regulatory element-binding protein (SREBP) family of transcription factors controls genes involved in cholesterol/lipid metabolism, membrane synthesis, and fat storage. Aberrant SREBP activity has been linked to conditions associated with metabolic syndrome, such as insulin resistance, obesity, elevated LDL-C and triglycerides, cardiovascular pathologies, and non-alcoholic fatty liver disease. Furthermore, SREBPs have been implicated in cancer cell proliferation and replication of enveloped viruses through regulation of membrane synthesis. We have previously defined the molecular requirements for gene activation by SREBPs, demonstrating a central role for the MED15 subunit of the Mediator co-activator complex and the CBP/p300 acetyltransferases. While most therapeutic approaches aimed at altering gene expression programs target upstream signaling pathways or nuclear translocation checkpoints, our studies have provided mechanistic insights supporting an alternative approach, which is aimed at directly inhibiting the interaction of the SREBP transactivation domains (TADs) with MED15 and CBP/p300 co-activators in the nucleus. The goal of the proposed research is to elucidate the mechanisms by which the SREBP transcriptional activators interact with co-activators, and to develop specific small-molecule inhibitors of these interactions. Through a combination of structural and biophysical methods in conjunction with high-throughput screening of small molecules, we propose to use structural insights to guide the development of new classes of therapeutics against cardiovascular and other diseases. In preliminary work, we have already been able to identify small-molecule inhibitors capable of disrupting SREBP-TAD binding to MED15, resulting in concomitant down-regulation of its target genes, such as fatty acid synthase (FASN). Inhibitors emerging from these studies will be optimized with medicinal chemistry. We will pursue the following specific aims: Specific Aim 1: Characterize structurally and functionally the SREBP interaction with co-activators MED15 and CBP/p300. Specific Aim 2: Discover and characterize in vitro small-molecule inhibitors of the SREBP-TAD interaction with co-activators to develop therapeutics against aberrant cholesterol/lipid metabolism. Specific Aim 3: Functionally characterize identified SREBP/co-activator inhibitors using both cell culture and mouse models.